Animal and cell models for type II diabetes and their use

ABSTRACT

A transgenic diabetes type II model laboratory animal is disclosed which comprises β-cells expressing a dominant negative form (dnFGFR1c) of FGFR1c. Also disclosed is the use of the Ipf1/Pdx1 promoter for controlling expression of FGFR1c; β-cells in wich the expression of PC1/3 is down-regulated or absent or which are competent to express a dominant negative form (dnFGFR1c) of FGFR1c; mature β-cells incompetent to express Glut2 or in which the processing of proinsulin is substantially impaired; a method of preventing or treating type II diabetes.

FIELD OF THE INVENTION

[0001] The present invention relates to an animal model for type IIdiabetes, in particular a transgenic mouse, in which the expression offibroblast growth factor receptors essential for ensuring a functionalβ-cell identity is perturbed. The invention also relates to the use ofthis model for studying type II diabetes, in particular with the aim ofdeveloping therapies therefore. The invention also relates to acorresponding cells and their components useful as in-vitro or in-vivomodels. Furthermore the invention relates to a method of preventing ortreating diabetes type-II.

BACKGROUND OF THE INVENTION

[0002] The Fibroblast Growth Factor (FGF) gene superfamily is a familyof conserved, secreted proteins that have been shown to play a criticalrole in many biological processes (Kato and Sekine, 1999; Szebenyi andFallon, 1999). FGF-signalling is achieved by binding of the ligand, FGF,to the extra-cellular domain of high affinity membrane bound FGFR, whichbelongs to the tyrosine kinase family of receptors (Kato and Sekine,1999; Szebenyi and Fallon, 1999). Today around 20 different FGF genesand 4 different FGFR genes have been identified, and multiple ligandscan interact with one and the same receptor (Kato and Sekine, 1999;Szebenyi and Fallon, 1999). The level of complexity of signalling viathese receptors is further compounded by the fact that alternativesplice variants exist for these receptors. Loop three of theextracellular domain (=ligand binding domain) can splice to give rise tob, or c, isoform. This isoform variation ultimately determines ligandspecificity and proper ligand-receptor interaction ultimately leads toactivation of the intracellular tyrosine kinase domain (Kato and Sekine,1999; Szebenyi and Fallon, 1999).

[0003] FGF-signalling has been implicated in a variety of distinctbiological processes including patterning, differentiation,morphogenesis, proliferation, survival, angiogenesis, tumorogenesis,etc. (Kato and Sekine, 1999; Szebenyi and Fallon, 1999). In mouse, anearly embryonic lethality or functional redundancy have, however,largely hampered direct genetic approaches aiming at elucidating therole of FGF-signalling during development and in the adult. Thus theseapproaches has for the most part failed to provide critical informationregarding the role of FGF-signalling during later stages of vertebrateorganogenesis, including the pancreas. An alternative approach have beento impair FGF-signalling via organ specific expression of dominantnegative forms of FGFR that will competitively block FGF signalling viathe endogenous, corresponding FGFR variant. This approach has beensuccessfully used to antagonise FGF-signalling in a number of differentsystems.

[0004] Viral infection of a dominant negative FGFR1 construct in chicklimb muscle mass blocked the differentiation of myoblast to myotubesproviding evidence that this process depends on FGF-signalling (Itoh etal., 1996). Studies focused on maintenance of cell types within theretina revealed that expression of dnFGFR2 under the control of thebovine rhodopsin promoter increased photoreceptor degeneration(Campochiaro et al. 1996). The specificity of dominant negativeconstructs with respect to ligand binding was demonstrated in analyseswhere dnFGFR1c and dnFGFR2b constructs where expressed in transgenicmice using the mammary tumour virus promoter (Jackson et al.,1997).Expression of dnFGFR1c under these conditions did not result in anydiscernible phenotype whereas an impairment of lobuloalveolardevelopment in the mammary gland was observed when using the dnFGFR2bvariant (Jackson et al.,1997). Moreover, FGF8 mediated induction ofdopaminergic (DA) neurons was successfully inhibited when growing sixsomite rat ventral mid/hindbrain explants in presence of solublednFGFR3c, i.e. the high-affinity blocking receptor for FGF8 (Ye et al.,1998). In contrast, when the same experiment was performed usingsoluble, dnGFGR1c, a low-affinity nonblocking receptor for FGF8, DAneurons readily appeared (Ye et al., 1998). Together these analysesdemonstrate the effectiveness by which FGF signalling, in an apparentligand-specific manner, can be perturbed using a dominant-negative FGFRapproach.

[0005] Three different FGF-signalling mutant mice, involving transgenicapproaches to over-express either a ligand or a dn form of a receptor,resulting in a pancreatic phenotypes have been reported. Transgenicover-expression of FGF7/KGF in the mouse liver induced pancreaticductual hyperplasia (Nguyen et al. 1996) and similarly, transgenic micewith forced expression of FGF-7/KGF in pancreatic β-cells under thecontrol of the insulin promoter show enlarged islets containingproliferating duct cells (Krakowski et al., 1999). General transgenicover-expression of dnFGFR2b under the control of the metallothioneinpromoter resulted in pancreatic hypoplasia (Celli et al. 1998). Togetherthese studies indicate that signalling through FGFR2b may operate duringpancreatic development. In vitro experiments involving culturing ofpancreatic rudiments support such a scenario and suggest that FGFspositively stimulate pancreatic epithelial cell proliferation andexocrine cell differentiation (Le Bras et al. 1998, Miralles et al.1999).

[0006] Selective inactivation of the IIIb form of FGFR2 leads todevelopmental abnormalities in limbs, lung, anterior pituitary, salivaryglands, inner ear, teeth and skin but apparently not in the pancreas (DeMoerlooze et al., 2000). Thus, the roles of FGFR2b during pancreasdevelopment remain to be determined.

[0007] Failure of the β-cell to compensate for an increased demand forinsulin is a key feature in the manifestation of type 2 diabetes. Type 2diabetes is the most common form of diabetes, affecting 2-3% of theworld-wide population, and is the combined result of resistance toinsulin action coupled with a defect in β-cell compensation (Kahn, 1998;Kahn and Rossetti, 1998; Taylor, 1999). The molecular defects underlyingthe development of the disease are not fully understood and there arealso uncertainties as to what is the primary defect initiating thedisease; the insulin resistance or the β-cell failure. A typical traitassociated with the disease is the increased proinsulin to insulin (P/I)ratio observed in many type 2 diabetic patients (Porte and Kahn, 1989).The relationship between the increased P/I ratio and the etiology of thedisease has however remained diffuse; i.e. is it a consequence ratherthan a directly contributing factor to the disease? Several independentstudies points towards an increased P/I ratio being an early sign ofprimary β-cell dysfunction, independent of insulin resistance, which isdirectly associated with the conversion from a prediabetic to an overtdiabetic state over a short time period (Mykkänen et al., 1995; Kahn etal., 1995, Nijpels et al., 1996; Rachman et al., 1997; Mykkänen et al.,1997; Haffner et al 1997; Larsson and Ahrén, 1999). Moreover, it hasbeen suggested that normal β-cells respond to an increased insulinresistance by enhanced processing of insulin and that the increased P/Iratio in individuals with an impaired glucose tolerance, and/or type 2diabetes, is the consequence of defects in proinsulin processing(Mykkänen et al., 1997, Larsson and Ahrén, 1999).

[0008] Processing of proinsulin to insulin in β-cells is catalysed bythe sequential actions of prohormone convertases PC1/3 and PC2, whichboth act in concert with carboxpeptidase E (CPE) (FIG. 7) Analyses ofPC2 null mutant mice demonstrated a crucial role for PC2 in theprocessing of proglucagon and prosomatostatin in α- and δ- cells,respectively (Furuta et al., 1998). Proinsulin processing in β-cells wasless affected in the PC2 null mutant mice providing evidence that PC3 isquantitatively more important than PC2 with respect to processing ofproinsulin to active insulin (Furuta et al., 1998). At present there isa lack of genetically defined animal models that mimic these aspects ofhuman type 2 diabetes. The importance of this disease in terms of humansuffering and health care costs makes the provision of such a model animportant goal.

OBJECTS OF THE INVENTION

[0009] It is an object of the present invention to provide an animalmodel which mimics human type II diabetes and which can be used todevelop a therapy.

[0010] It is another object of the invention to provide a method ofpreventing or treating type II diabetes.

[0011] Further objects of the invention will become evident from thestudy of the following short description of the invention and preferredembodiments thereof, the figures illustrating the invention, and theappended claims.

SUMMARY OF THE INVENTION

[0012] The invention is based on the insight that, in addition to thepreviously reported expression of FGFR2b during pancreas development,FGFR2 and FGFR1 are both expressed in the adult β-cell. In regard ofFGFR1, a functional role was demonstrated by impairing signallingthrough FGFR1c via the expression of a dominant negative form of thisreceptor, dnFGFR1c (Ye et al., 1998), under control of the Ipf1/Pdx1promoter (Apelqvist et al., 1997).

[0013] Mice expressing the dnFGFR1c exhibit a grossly normally developedpancreas with no apparent abnormalities but develop diabetes with age.The expression of dnFGFR1c in β-cells results in disorganised isletswith reduced numbers of β-cells displaying an apparent immaturemolecular identity. First, the β-cells do not express detectable oflevels Glut2 which is one of the key components of the glucose sensingmachinery. Secondly, the expression of one of the proinsulin processingenzymes, PC1/3, is impaired. Third, although insulin is synthesised bythe β-cells it fails to be fully processed and remains largely in theform of pro-insulin and/or partially processed.

[0014] According to the present invention, evidence is provided that theexpression of FGFR1 is dependent on Ipf1/Pdx1 expression. Rip1/Ipf1⁵⁶⁸mice in which Ipf1/Pdx1 has been inactivated selectively in β-cellsdisplay disorganised islets and develop diabetes due to decreasedinsulin expression combined with a loss of Glut2 expression (Ahlgren etal., 1998). The expression of both FGFR 1 is down-regulated in theRip1/Ipf1^(▴) mice suggesting that Ipf/Pdx1 is required for expressionof FGFR1 and FGFR2 in β-cells. Moreover, in the Rip1/Ipf1^(▴) mice,alike in the Ipf1/dnFGFR1c mice, PC1/3 expression is impaired. Theseresults suggest that signalling via FGFR1c is required for ensuring acorrect number of β-cells and their proper function. Moreover, theresults provide evidence that Ipf1/Pdx1 controls many aspects of theβ-cell glucose homeostasis machinery, in part, by being required for theexpression of FGFR1 in the β-cell.

[0015] In humans, heterozygosity for a nonsense mutation in the Ipf1gene, which results in a dominant negative frameshift, has been linkedto Maturity-Onset Diabetes of the Young (MODY) 4 [Stoffers et al.,1997], a monogenetic form of diabetes that results from β-celldysfunction rather than insulin resistance. Moreover, missense mutationsin the human Ipf1 gene are implicated in predisposing an individual totype 2 diabetes [Macfarlane et al., 1999; Hani et al., 1999]. Previouswork has shown that Ipf1/Pdx1 is required for ensuring normal levels ofinsulin and Glut2 expression [Ohlsson et al., 1993; Ahlgren et al.,1998]. According to the present invention, as explained above, there isnow genetic evidence suggesting that Ipf1/Pdx1 acts upstream ofFGF-signalling in the β-cell, since genetic inactivation of Ipf1/Pdx1 inβ-cells, as in the RIP1/Ipf1^(Δ) mice [Ahlgren et al., 1998], leads toreduced expression levels of FGFR1, and the ligands FGF1, FGF2, FGF4 andFGF5. Consequently, the RIP1/Ipf1^(Δ) mice also display reducedexpression of PC1/3 paralleled by an increase in proinsulin in theβ-cells of these mice.

[0016] The phenotypes observed in the FRID1 mice, i) reduced β-cellnumber, ii) loss of Glut2 expression leading to impaired glucose sensingand, iii) perturbed proinsulin processing due to the down regulation ofprohormone convertase ⅓ and 2 expression [6], are reflective of theβ-cell dysfunction associated with type 2 diabetic patients [Porte etal., 1989; Hales, 1994; Mykkanen et al., 1995; Mykkanen et al., 1999;Kahn et al., 1995; Kahn et al., 1995 bis; Larsson et al., 1999; Nijpelset al., 1996; Rachman et al., 1997; Haffner et al., 1997]. Thesefindings suggest that signalling via FGFR1c may represent one factorrequired for β-cell expansion both during early life and in response tohyperglycaemia. Morover these data provide evidence thatFGFR1c-signalling in the β-cell is required to ensure normal expressionof key components in glucose sensing (Glut2) and insulin processingmachinery (PC1/3 and PC2) and thus to maintain normoglycaemia. Last theanalyses of the RIP1/Ipf1^(Δ) mice provide genetic evidence that thatthe IPF1/PDX1 transcription factor acts upstream of FGFR1-signalling incontrolling key aspects of β-cell identity. The apparent conservation ofIpf1/Pdx1 gene function from mice to humans suggest that also thedownstreams effects controlled by Ipf1/Pdx1 gene activity may beconserved [Stoffers et al., 1997; Macfarlane et al, 1999; Hani et al.,1999; Ohlsson et al., 1993; Ahlgren et al., 1998]. This stronglysuggests that that FGF-signalling is important for β-cell function alsoin humans and that pertubation of this signalling pathway in adult humanβ-cells is linked to type II diabetes.

[0017] According to the present invention thus is disclosed a transgenicdiabetes type II model laboratory animal comprising β-cells expressing adominant negative form (dnFGFR1c) of FGFR1c. In particular thetransgenic animal is a mouse.

[0018] According to the present invention is also disclosed the use ofthe Ipf1/Pdx1 promoter for controlling the expression of FGFR1c.

[0019] According to a first preferred aspect of the invention aredisclosed β-cells in which the expression of PC1/3 is down-regulated orabsent. Preferably the β-cells are comprised by an adult pancreas.

[0020] According to a second preferred aspect of the invention aredisclosed β-Cells competent to express a dominant negative form(dnFGFR1c) of FGFR1c. Preferably the β-cells are comprised by an adultpancreas.

[0021] According to a third preferred aspect of the invention aredisclosed mature β-cells incompetent to express Glut2. Preferably theβ-cells are comprised by an adult pancreas.

[0022] According to a fourth preferred aspect of the invention aredisclosed mature β-cells in which the processing of proinsulin toinsulin is substantially impaired. In these cells levels of proinsulinconvertase ⅓ are substantially reduced in comparison with the levels innon-transgenic mice. Preferably these β-cells are comprised by an adultpancreas.

[0023] According to a fifth preferred aspect of the invention aredisclosed a reduced number of β-cells and a failure of β-cells torespond to hyperglycemia by replication.

[0024] In the following the invention will be by described in moredetail by reference to preferred but not limiting embodiments.

SHORT DESCRIPTION OF THE FIGURES

[0025] The preferred embodiments are illustrated by a number of figuresshowing:

[0026]FIG. 1 expression of FGFR1 in adult β-cells;

[0027]FIG. 2 reduction in number of ins⁺-cells in Ipf1/dnFGFR1c mice;

[0028]FIG. 3 defects in β-cell identity in Ipf1/dnFGFR1c mice;

[0029]FIG. 4 impaired pro-insulin processing in Ipf1/dnFGFR1c mice;

[0030]FIG. 5 control by pf1/Pdx1 of multiple aspects of β-cell identityincluding FGFR1 expression.

DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1 Expression of FGFR1 inthe Adult Pancreas

[0031] Analysis of FGFR1 expression in the adult mouse pancreas revealedthat FGFR1 is predominantly expressed in the adult β-cell, with noexpression observed in the glucagon-producing α-cells (FIG. 1). A lowerlevel of FGFR1 expression was also observed in the exocrine cells of thepancreas (data not shown). FGFR2 is also selectively expressed in theadult β-cells but in contrast to FGFR1, FGFR2 expression was notobserved in the exocrine cells of the adult pancreas (data not shown).The expression of FGFR in adult β-cells suggests a role forFGF-signalling in terminal differentiation and/or maturation of thesecells.

[0032] The expression of FGFR1 in the pancreas led us to examine whethersignalling via this receptor may be required for pancreas development,as has already been implied for FGFR2b. We also examined whether FGFreceptor signalling was required for the specification anddifferentiation of adult β-cells. To begin to address this issue wegenerated transgenic mice expressing dominant negative FGFR1c constructin the pancreas using the Ipf1/Pdx1 promoter. This consisted of thethree loop extracellular domain of FGFR1c fused in frame with the ratIgG Fc region. In the resulting mice Ipf1-expressing cells would secretethe hybrid protein from the cell allowing competitive binding of FGFligands such as FGF1, FGF2, FGF4, FGF5 and FGF6 which are known to bindto the FGFR1c variant.

EXAMPLE 2 Development Diabetes in Ipf1/dnFGFR1c Mice.

[0033] Ipf1/dnFGFR1c express high levels of the transgene in β-cellswith lower levels of the transgene being expressed in the exocrine partof the pancreas (FIG. 1 and data not shown). The Ipf1/dnFGFR1ctransgenic mice are viable and fertile with a grossly well developedpancreas, providing evidence that signalling via FGFR1c is not importantfor pancreas growth, morphogenesis and differentiation. The miceappeared healthy until approximately 15 weeks of age when elevatednon-fasting urine glucose concentrations greater than 2% were observedsuggesting a diabetic phenotype. Fasting blood glucose measurements onmice were >20 mM, confirming that these animals were severely diabetic.

[0034] Close monitoring of urine and blood glucose levels revealed thatalready at 3 weeks of age Ipf1/dnFGFR1c mice showed elevated glucoselevels, albeit still within the normal range (Table 1). At six weeks ofage their fasting blood glucose levels had increased by 1 mM and theyhad detectable levels of glucose in their urine when compared to wildtype age matched littermates (Table 1). Weekly monitoring of the urineglucose revealed a steady increase in glucose and by the age of 9-12weeks urine glucose levels were in excess of 2%. Non-fasting and fastingblood glucose measurements taken at this stage revealed 4-fold higherglucose levels compared with age-matched wild type littermates (Table1). These findings demonstrate that impaired FGFR1c-signaling in adultβ-cells results in the development of diabetes and points to a crucial,hitherto unknown role for FGF-signalling in β-cell glucose homeostasis.TABLE 1 Ipf1/dnFGFR1c transgenic mice develop diabetes Blood glucoselevels (mM +/− S.E.M.) non-fasted fasted Wild type (3 weeks old) nd 4.4+/− 0.4 (n = 5) Ipf1/dnFGFR1c (3 weeks old) nd 5.8 +/− 0.5 (n = 7) Wildtype (6 weeks old) nd 4.9 +/− 0.3 (n = 5) Ipf1/dnFGFR1c (6 weeks old) nd6.8 +/− 0.8 (n = 7) Wild type (12 weeks old)  8.7 +/− 0.8 (n = 5) 4.0+/− 0.6 (n = 5) Ipf1/dnFGFR1c 26.4 +/− 1.5 (n = 5) 15.8 +/− 1.3 (n = 7)(12 weeks old)

[0035] Legend to Table 1: Blood glucose levels were measured inIpf1/dnFGFR1c mice and wild type littermates at the time points shown.At 3 weeks of age Ipf1/dnFGFR1c mice showed non-fasted blood glucoselevels within the normal range. Six-week old Ipf1/dnFGFR1c mice hadslightly elevated non-fasted blood glucose levels, still within thenormal range albeit at the upper level. Overt diabetes (OD) develops in9-12 weeks old mice in whom both non-fasted and fasted blood glucoselevels were 4-fold higher than wild type, age-matched littermates.

EXAMPLE 3 Demonstration of Disorganized Islets with Reduced Numbers ofβ-cells in Ipf1/dnFGFR1c Mice

[0036] The overtly normal development of the pancreas in Ipf1/dnFGFR1cmice homozygous mutants suggested that the growth and differentiation ofthe pancreas is independent of FGFR1c-signaling. To assess this furtherwe analyzed the expression of the transcription factors Isl1, Ipf1/Pdx1,Nkx6.1 and Nkx2.2, the endocrine hormones insulin (Ins), glucagon (Glu),somatostatin (Som), and the exocrine enzymes amylase andcarboxypeptidase A. Each of these markers were expressed in the pancreasof Ipf1/dnFGFR1c mice and the organization of endocrine cells intoislet-like clusters and of exocrine cells into acinar-like structuresappeared normal (Data not shown). Nevertheless, as revealed by doubleimmunohistochemical analysis, there was a 35% decrease in the totalnumber of Isl1⁺ cells paralleled by ˜30% net decrease in the number ofIns⁺-cells and a concomitant 20% increase in the relative number ofGlu⁺-cells (FIG. 2). These results suggest that the genesis and/orsurvival of β-cells partly depends on FGFR1c-signalling. Tunnel assaysfailed, however, to detect any increased β-cell apoptosis suggestingthat the decreased number of β-cells are not caused by β-cell death(data not shown). The 30% decrease in total number of insulin cells inthe transgenic mice was reflected by a 28% decrease in total pancreaticinsulin content (FIG. 2). Moreover, although islets form in theIpf1/dnFGFR1c mice the typical structure of maturing islets with α-cellsat the periphery surrounding a core of β-cells is perturbed; instead,Glu⁺ cells are found scattered throughout the islets (FIG. 3). Incombination these histological analyses support the idea that thedevelopment of the pancreas to generate both exocrine and endocrinecells is unaffected despite the expression of a dn form of FGFR1c duringpancreas development and in the adult β-cell. In addition these resultsprovide evidence of the genesis of pancreatic β-cells appearing to bepartly dependent on, and that normal organization of islet-cellsrequires, FGFR1c-signaling.

EXAMPLE 4 Down-Regulation of Glucose Transporter Type 2 is inIpf1/dnFGFR1c Mice

[0037] The decrease in total insulin production by 28% appears unlikelyto be sufficient to cause the diabetes observed in the transgenic miceand suggests that additional complications underlie the development ofthe diabetic phenotype observed in the Ipf1/dnFGFR1c mice. To determinewhether other key characteristics of the adult β-cells were affected inthe transgenic mice we next monitored the expression of factorscrucially required for normal glucose homeostasis. Glut2 is a keycomponent in glucose sensing machinery within the β-cell. Analysesrevealed that the expression of Glut2 was virtually lost in overtdiabetic Ipf1/dnFGFR1c mice (FIG. 3). To exclude that the loss of Glut2expression was a consequence of the hyperglycemic state rather than adirect effect of the Ipf1/dnFGFR1c transgene expression, 5-week old,prediabetic transgenic mice were analyzed. Prediabetic, 5 week oldIpf1/dnFGFR1c mice exhibit a clearly reduced level of Glut2 expressionas compared to wild type (not shown). These results indicate that thereduction of Glut2 expression observed in Ipf1/dnFGFR1c mice is a directconsequence of impaired FGFR1c signalling.

EXAMPLE 5 Down-Regulation of PC1/3 in Ipf1/dnFGFR1c Mice

[0038] Although the loss of Glut2 may be sufficient to cause theirdiabetic phenotype, the severity of the ensuing hyperglycemia in a shortperiod of time suggested that there might be additional defects in theβ-cell of Ipf1/dnFGFR1c mice. Type 2 diabetes patients and animal modelsof the disease, often suffer from hyperproinsulinemia, reflecting animpaired processing of proinsulin to mature, active insulin which isbelieved to be a major contributing factor to their disease. Toelucidate a potential processing defect in the Ipf1/FGFR1dn mice weperformed an immunohistochemical analysis for investigating theexpression of the proinsulin processing enzymes, PC1/3 and PC2. PC1/3expression was found to be severely down-regulated so as to be virtuallyabsent in the Ipf1/dnFGFR1c mice, whereas only a minor decrease wasobserved with respect to PC2 expression in overt diabetic mice (FIG. 3and data not shown).

[0039] Next we wanted to determine whether this aberrant processingenzymes expression could be directly involved in the development of thediabetic phenotype in the Ipf1/dnFGFR1c mice. To this effect pancreasfrom five-week old prediabetic and wild type littermates were analyzedfor PC1/3 and PC2 expression. The analyses showed that already at5-weeks of age expression levels of the prohormone convertases wasreduced in the Ipf1/dnFGFR1c mice as compared to controls (not shown).Together, these results suggest that FGFR1c signaling is required forhigh level expression of PC1/3, and to a lesser extent for PC2expression.

EXAMPLE 6 Demonstration of impaired Proinsulin Processing inIpf1/dnFGFR1c Mice

[0040] To address whether the impaired expression of the processingenzymes might affect insulin processing in the Ipf1/dnFGFR1c mice weexamined the type of insulin made and stored in the β-cells. An antibodydirected against intact human proinsulin, which do not cross-react withactive insulin (Madsen et al., 1983; Madsen et al., 1984; Furuta et al.,1998), and antibodies directed against mouse C-peptide 1 and 2 (Blume etal., 1992) were used to evaluate the forms of insulin present in theβ-cells of Ipf1/dnFGFR1c mice.

[0041] There was a marked reduction in the levels of both C-peptide 1and C-peptide 2 in the β-cells of the overt diabetic transgenic mice ascompared to β-cells of wild type mice (FIG. 4). In contrast, high levelsof proinsulin was observed in the β-cells of the transgenic mice whileno or very little proinsulin was observed in the wild type β-cells (FIG.4). Notably increased proinsulin levels were manifest already at theprediabetic 5-week stage (data not shown). These results indicate thatthe down regulation of the processing enzyme, PC1/3 in the Ipf1/dnFGFR1cmice results in an impaired processing of proinsulin to its activemature form. This processing defect is likely to contribute to thedevelopment of severe diabetes in the Ipf1/dnFGFR1c mice. Thus impairedsignaling via FGFR1c in the adult β-cell leads to both aberrant glucosesensing and impaired insulin processing that together ultimatelyprogress to diabetes development.

EXAMPLE 7 Demonstration that Ipf1/Pdx1 is Required for the Expression ofFGFR1 and FGFR2

[0042] The disorganisation of islets and the down-regulation of Glut2observed in the Ipf1/dnFGFR1c mice resembles the islet phenotypeassociated with a β-cell specific activation of the transcription factorIpf1/Pdx1 demonstrated in the RIP1^(Δ)/Ipf1 mice previously establishedin our laboratory (Ahlgren et al., 1998). These mice develop diabetes at10-15 weeks of age due to a Cre-mediated excision exon 2 of theIpf1/Pdx1 gene. Analysis of pancreas derived from RIP1^(Δ)/Ipf1 miceshowed that the expression of FGFR1 and FGFR2 was down-regulated in theβ-cells (FIG. 5 and data not shown). Some residual expression couldstill be observed which probably reflects a residual Ipf1 expression insome of the β-cells, since these mice develop diabetes at a stage whenapproximately 20% of the β-cells still express Ipf1 (Ahlgren et al.,1998). To investigate whether the decrease in FGFR1c expression in theRIP1^(Δ)/Ipf1 mice might lead to a perturbed insulin processing weperformed an analysis of the expression of the prohormone convertases inthe RIP1^(Δ)/Ipf1 mice. PC1/3 was severely down-regulated in these miceas well (FIG. 5). The RIP1^(Δ)/Ipf1 mice also displayed an increasedproinsulin content in their β-cells coupled with a decreased levels ofC-peptide 1 and 2 (FIG. 5, and data not shown).

[0043] In combination these results provide evidence that Ipf1 isrequired for the expression of FGFR1 in the adult β-cells, therebyensuring a high level of Glut2 and PC1/3 expression. Thus, bothperturbed Ipf1 expression and impaired signalling via FGFR1c leads todiabetes due to impaired glucose sensing and insulin processing in theadult β-cells with diabetes manifestation as a consequence. Theseresults suggest that signalling via FGFR1c is critical for themaintenance of a mature, functional β-cell phenotype, and that Ipf1/Pdx1by virtue of its key role in controlling, directly or indirectly, manyaspects of the β-cell glucose homeostasis machinery is pivotal for theβ-cell's capacity to preserve normoglycemia.

EXAMPLE 8 Analysis of Pancreas from Diseased Type 2 Diabetics andControl Individuals

[0044] Double immunohistochemical analyses of pancreas derived formnon-diabetic humans show that both FGFR1 and FGFR2 are selectivelyexpressed also in -human insulin-producing β-cells and not glucagonproducing α-cells (FIG. 6a and 6 b). Similar to mice with diabetes dueto impaired FGFR1-signalling [5,6], type 2 diabetics displaydisorganized islets with glucagon-producing α-cells mixed with theinsulin-producing β-cells (FIG. 6d), whereas non-diabetics have normalislets with glucagon-producing cells surrounding the core ofinsulin-producing β-cells (FIG. 6c).

[0045] Moreover, the expressions of both FGFR1 and FGFR2 are drasticallyreduced in the insulin-producing β-cells of type 2 diabetics as comparedto that of control individuals (FIG. 7). Consequently, as has been shownfor mice with impaired FGFR1 signalling, type 2 diabetics show reducedexpression of PC1/3 in their β-cells (FIG. 8b, 8 e) whereas theexpression of PC2 appear less affected (FIG. 8a, 8 d). The reduction ofPC1/3 expression is paralleled by an increase in β-cell proinsulincontent in the Type 2 diabetics as compared to the control individuals(FIG. 8c, 8 f).

[0046] These experiments demonstrate that β-cells of type 2 diabeticpatients have disorganized islets, reduced expression of FGFR1, FGFR2and PC1/3 as well as an increased proinsulin content in their β-cells.These findings when combined provide evidence that, similar to mice withdiabetes due to an impaired FGFR1-signalling in adult β-cells,attenuation of FGFR1-signalling pathway in human β-cells is coupled todiabetes. We suggest that FGF-signalling in the adult pancreas ensures afunctional β-cell identity and glucose homeostasis. Thus an impairedexpression, or activity, of components within the FGF-signalling pathwayis coupled to diabetes in both mice and humans. In both mice and humansimpaired FGF-expression and signalling is coupled to a decrease in PC1/3expression. The decrease in PC1/3 in turn leads to a perturbedprocessing of insulin with elevated pro insulin levels as a result.Consequently the FGF-signalling pathway, i.e. including componentsupstream and downstream of the FGFR-ligand interaction, is a suitabletarget for the development of new therapies to cure diabetes.

[0047] We have also recent data from analyses of expression ofId-proteins [Norton, 2000] that Id2 and Id3 are targets downstream ofFGFR1-signalling. As is shown in FIG. 9, both Id2 and Id3 are normallyexpressed in pancreatic endocrine cells (FIG. 9a, 9 d) . However, inmice with diabetes due to attenuation of FGF-signalling in β-cells, i.e.the FRID1 and RIP1/Ipf1^(Δ) mice, the expression of both Id2 (FIG. 9b, 9c) and Id3 (FIG. 9e, 9 f) are severely down-regulated. These resultsprovide evidence that Id2 and Id3 expression in β-cells are dependent ofFGF-signalling and hence represent downstream components of theFGF-signalling pathway in adult β-cells. Id2 and Id3 thus representcandidate targets for the development of new therapies to cure diabetes.

[0048] FIGURE LEGENDS

[0049]FIG. 1: (A-F) Analysis of FGFR1 expression in adult pancreasshowing that FGFR1-expression (B,D) coincides with insulin (C) but notglucagon (A) expression. (E,F) In Ipf1/dnFGFR1c transgenic mice thednFGFR1c protein (F) is highly expressed in IPF1⁺ cells (G) as detectedby antibodies against the Rat IgG Fc region that is coupled to thednFGFR1c domain.

[0050]FIG. 2: (A) Analysis of number Ins⁺-cells over number ofIsl1⁺-cells showing a 30% decrease in the number of Ins⁺-cells inIpf¹/dnFGFR¹c (TG) mice as compared to wild-type (wt) mice. Data from atleast 4 independent mice, n=7435 cells. (B) Measurement of totalpancreatic insulin content (μg insulin/mg pancreas protein) fromnon-fasted animals (n=6 wt and n=8 TG) show that the 30% decrease innumber of Ins⁺-cells (A) is accompanied by a 28% decrease in totalpancreatic insulin content in the Ipf1/dnFGFR1c mice as compared withtheir age-matched wild type littermates. (C) The 30% decrease in numberof insulin cells results in a relative 20% increase in number ofGlu⁺-cells in Ipf¹/dnFGFR¹c mice. Data from at least 4 independent mice,n=7525 cells.

[0051]FIG. 3: (A-D) Confocal microscopy analyses of insulin (C,D) andglucagon (A,B) expression in wild-type (A,C) and Ipf¹/dnFGFR1ctransgenic (B,D) mice show that the islet organisation in the transgenicmice is abnormal but that there is no co-expression between insulin andglucagon within the islet cells. (E-H) Expression of glucose sensing andinsulin processing enzymes is impaired in Ipf1/dnFGFR1c mice. Imagesshow that Glut2-expression (E,F) in adult β-cells is lost as a result ofIpf1/dnFGFR1c expression and that only a very low level of PC3expression (G,H) remains in Ipf1/dnFGFR1c mice, and preferentially inα-cells.

[0052]FIG. 4: Analyses of insulin variants in pancreas from wild-type(A, C, E, G) and Ipf1/dnFGFR1c (B, D, F, H) mice using C-peptide 1 (Aand B), C-peptide 2 (C and D), insulin (E and F) and proinsulin (G andH) anti-sera. (A, C, E, G) In wild-type pancreas insulin is presentpredominantly in its fully processed form (A, C, E) with virtually nodetectable unprocessed pro-insulin (G). Note that the fluorescence in Crepresents background, non-β-cell reactivity due to the use of mousemonoclonal anti-sera. (B, D, F, H) Ipf1/dnFGFR1c mice display aperturbed proinsulin processing resulting in reduction of fullyprocessed insulin (B, D, F) while a substantial, readily detectablefraction remains in the form of proinsulin (H).

[0053]FIG. 5: (A-F) Loss of IPF1/PDX1 activity in β-cells (Ahlgren etal. 1998) results in a drastically reduced FGFR1 (A,B) and PC3 (C,D)expression. This loss of PC3 expression results in perturbed proinsulinprocessing, with a reduction in detectable levels of fully processedinsulin (not shown) accompanied by increased levels of detectableproinsulin (E,F) within the β-cells of the Rip1/Ipf1^(Δ) mice (B,D,F) ascompared to wild-type littermates (A,C,E).

[0054]FIG. 6: Expression of FGFR1 and FGFR2 in adult human islets.Confocal microscopy analyses of FGF receptor and glucagon expression inadult human pancreas showing that the receptors FGFR1 (a) and FGFR2 (b)are expressed in □-cells and not in β-cells. The islets of human type 2diabetic patients (n=3) (d) are disorganized with glucagon producingcells found scattered throughout the islets, as compared withnon-diabetic pancreatic tissue where the glucagon cells are found at theperiphery of the islet (c).

[0055]FIG. 7: Impaired expression of FGFR1 and FGFR2 in human type 2diabetic patients. Immunohistochemical analyses demonstrate that bothFGFR1 (a and c) and FGFR2 (b and d) are expressed at clearly reducedlevels in β-cells of patients (n=3) suffering from type 2 diabetes.Non-diabetic pancreatic tissue (a and b), diabetic pancreatic tissue (cand d).

[0056]FIG. 8: Impaired expression of PC1/3 in human type 2 diabeticpatients. Immunohistochemical analyses show that PC2 (a and d)expression is unaffected in human type 2 diabetic patients (n=3),whereas expression of PC1/3 (b and e) is drastically reduced. Thedown-regulation of PC1/3 expression leads to functional impairment ofinsulin processing as revealed by the increase in intracellularproinsulin content (c and f). Non-diabetic pancreatic tissue (a-c),diabetic pancreatic tissue (d-f).

[0057]FIG. 9: Id2 and Id3 are downstream of FGFR1c-signalling. Id2 andId3 down-regulated in β-cells of overt diabetic FRID1 and RIP1/Ipf1^(Δ)mice. Both Id2 and Id3 are normally expressed in mouse adult isletscells (a,d). The expression of Id2 (a-c) is greatly reduced in β-cellsof diabetic FRID1 (b,e) and RIP1/Ipf1^(Δ) (e,f) mice as compared to thatof wild type littermates and the expression of Id3 (d-f) virtuallyabsent (e,f) in these diabetic mouse models. The remaining Id2 and Id3expression still observed in both the FRID1 and RIP1/Ipf1^(Δ) isletsrepresent expression in scattered glucagon cells.

[0058] REFERENCES

[0059] Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H (1998).β-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in lossof the beta-cell phenotype and maturity onset diabetes. Genes Dev 12,1763-8.

[0060] Apelqvist A, Ahlgren U, Edlund H (1997). Sonic hedgehog directsspecialised mesoderm differentiation in the intestine and pancreas. CurrBiol 7:801-4.

[0061] Blume N, Petersen J S, Andersen L C, Kofod H, Dyrberg T,Michelsen B K, Serup P, Madsen O D. (1992). Immature transformed isletb-cells differentially express C-peptides derived from genes coding forinsulin I and II as well as a transfected human insulin gene. MolEndocrinol 6:299-307.

[0062] Campochiaro P A, Chang M, Ohsato M, Vinores S A, Nie Z,Hjelmeland L, Mansukhani A, Basilico C, Zack D J (1996). Retinaldegeneration in transgenic mice with photoreceptor-specific expressionof a dominant-negative fibroblast growth factor receptor. J Neurosci 16,1679-88.

[0063] Celli G, LaRochelle W J, Mackem S, Sharp R, Merlino G (1998).Soluble dominant-negative receptor uncovers essential roles forfibroblast growth factors in multi-organ induction and patterning. EMBOJ 17, 1642-55.

[0064] De Moerlooze L, Spencer-Dene B, Revest J, Hajihosseini M,Rosewell I, Dickson C (2000). An important role for the IIIb isoform offibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelialsignalling during mouse organogenesis. Development 127, 483-92.

[0065] Furuta M, Carroll R, Martin S, Swift H H, Ravazzola M, Orci L,Steiner D F (1998). Incomplete processing of proinsulin to insulinaccompanied by elevation of Des-31,32 proinsulin intermediates in isletsof mice lacking active PC2. J Biol Chem 273:3431-7

[0066] Haffner S M, Gonzalez C, Mykkanen L, Stern M (1997). Totalimmunoreactive proinsulin, immunoreactive insulin and specific insulinin relation to conversion to NIDDM: the Mexico City Diabetes Study.Diabetologia, 40:830-7.

[0067] Hani E H, Stoffers D A, Chevre J C, Durand E, Stanojevic V, DinaC, Habener J F, Froguel P (1999). Defective mutations in the insulinpromoter factor-1 (IPF-1) gene in late-onset type 2 diabetes mellitus. JClin Invest 104:R41-48.

[0068] Hales C N (1994). The pathogenesis of NIDDM. Diabetologia 37Suppl 2, S162-8.

[0069] Itoh N, Mima T, Mikawa T (1996). Loss of fibroblast growth factorreceptors is necessary for terminal differentiation of embryonic limbmuscle. Development, 122, 291-300.

[0070] Kahn B B (1998). Type 2 diabetes: when insulin secretion fails tocompensate for insulin resistance. Cell 92:593-6.

[0071] Kahn B B, Rossetti L (1998). Type 2 diabetes—who is conductingthe orchestra? Nat Genet 20:223-5.

[0072] Kahn S E, Leonetti D L, Prigeon R L, Boyko E J, Bergstrom R W,Fujimoto W Y (1995). Proinsulin as a marker for the development of NIDDMin Japanese-American men. Diabetes 44:173-9. Kahn S E et al. (1995).Proinsulin as a marker for the development of NIDDM in Japanese-Americanmen. Diabetes 44: 173-179.

[0073] Kahn S E et al. (1995). Relationship of proinsulin and insulinwith noninsulin-dependent diabetes mellitus and coronary heart diseasein Japanese-American men: impact of obesity—clinical research centerstudy. J Clin Endocrinol Metab 80: 1399-1406.

[0074] Kato S, Sekine K (1999). FGF-FGFR signaling in vertebrateorganogenesis. Cell Mol Biol 45:631-8.

[0075] Krakowski M L, Kritzik M R, Jones E M, Krahl T, Lee J, Arnush M,Gu D, Sarvetnick N (1999). Pancreatic expression of keratinocyte growthfactor leads to differentiation of islet hepatocytes and proliferationof duct cells. Am J Pathol 154:683-91.

[0076] Larsson H, Ahren B (1999). Relative hyperproinsulinemia as a signof islet dysfunction in women with impaired glucose tolerance. J ClinEndocrinol Metab 84:2068-74.

[0077] Le Bras S, Miralles F, Basmaciogullari A, Czernichow P,Scharfmann R (1998). Fibroblast growth factor 2 promotes pancreaticepithelial cell proliferation via functional fibroblast growth factorreceptors during embryonic life. Diabetes 47, 1236-42.

[0078] Macfarlane W M, Frayling T M, Ellard S Evans J C, Allen L I,Bulman M P, Ayres S, Shepherd M, Clark P, Millward A, Demaine A, WilkinT, Docherty K, Hattersley A T (1999). Missense mutations in the insulinpromoter factor-1 gene predispose to type 2 diabetes. J Clin Invest104:R33-39.

[0079] Madsen O D, Cohen R M, Fitch F W, Rubenstein A H, Steiner D F(1983). The production and characterization of monoclonal antibodiesspecific for human proinsulin using a sensitive microdot assayprocedure. Endocrinology 113: 2135-2144.

[0080] Madsen O D, Frank B H, Steiner D F (1984). Humanproinsulin-specific antigenic determinants identified by monoclonalantibodies. Diabetes 33:1012-1016.

[0081] Miralles F, Czernichow P, Ozaki K, Itoh N, Scharfmann R (1999).Signaling through fibroblast growth factor receptor 2b plays a key rolein the development of the exocrine pancreas. Proc Natl Acad Sci U S A96, 6267-72.

[0082] Mykkänen L, Haffner S M, Kuusisto J, Pyorala K, Hales C N, LaaksoM (1995). Serum proinsulin levels are disproportionately increased inelderly prediabetic subjects. Diabetologia 38:1176-82.

[0083] Mykkänen L, Haffner S M, Hales C N, Ronnemaa T, Laakso M (1997).The relation of proinsulin, insulin, and proinsulin-to-insulin ratio toinsulin sensitivity and acute-insulin-response in normoglycemicsubjects. Diabetes 46:1990-5.

[0084] Mykkanen L, Zaccaro D, Hales C N, Festa A, Haffner S M (1999).The relation of proinsulin and insulin to insulin sensitivity and acuteinsulin response in subjects with newly diagnosed type II diabetes: theInsulin Resistance Atherosclerosis Study. Diabetologia 42:1060-1066.

[0085] Nguyen H Q, Danilenko D M, Bucay N, DeRose M L, Van G Y, ThomasonA, Simonet W S (1996). Expression of keratinocyte growth factor inembryonic liver of transgenic mice causes changes in epithelial growthand differentiation resulting in polycystic kidneys and other organmalformations. Oncogene 12, 2109-19.

[0086] Nijpels G, Popp-Snijders C, Kostense P J, Bouter L M, Heine R J(1996). Fasting proinsulin and 2-h post-load glucose levels predict theconversion to NIDDM in subjects with impaired glucose tolerance: theHoorn Study. Diabetologia 39:113-8.

[0087] Norton J D (2000). ID helix-loop-helix proteins in cell growth,differentiation and tumorigenesis. J Cell Sci. 113:3897-3905.

[0088] Ohlsson H, Karlsson K, Edlund T (1993). IPF1, ahomeodomain-containing transactivator of the insulin gene. Embo J 12:4251-4259.

[0089] Porte D Jr, Kahn S E. Hyperproinsulinemia and amyloid in NIDDM(1989). Clues to etiology of islet beta-cell dysfunction? Diabetes38:1333-6.

[0090] Rachman J, Levy J C, Barrow B A, Manley S E, Turner R C (1997).Relative hyperproinsulinemia of NIDDM persists despite the reduction ofhyperglycemia with insulin or sulfonylurea therapy. Diabetes46:1557-1562.

[0091] Stoffers D A, Ferrer J, Clarke W L, Habener J F (1997).Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet17:138-139.

[0092] Szebenyi G, Fallon J F (1999). Fibroblast growth factors asmultifunctional signaling factors. Int Rev Cytol 185:45-106.

[0093] Taylor S I (1999). Deconstructing type 2 diabetes. Cell 97:9-12.

[0094] Ye W, Shimamura K, Rubenstein J L, Hynes M A, Rosenthal A (1998).FGF and Shh signals control dopaminergic and serotonergic cell fate inthe anterior neural plate. Cell 93, 755-66.

1. A transgenic diabetes type II model laboratory animal comprisingβ-cells expressing a dominant negative form (dnFGFR1c) of FGFR1c.
 2. Thetransgenic animal of claim 1 or 2, wherein the animal is a mouse.
 3. Useof the Ipf1/Pdx1 promoter for controlling expression of FGFR1c. 4.β-Cells having been genetically modified so that the expression of PC1/3is down-regulated or absent.
 5. β-Cells having been genetically modifiedto express a dominant negative form (dnFGFR1c) of FGFR1c.
 6. The β-cellsof claim 4 or 5 comprised by an adult pancreas.
 7. The β-cells of claim5 or 6, wherein the β-cells are incompetent to express Glut2.
 8. Theβ-cells of claim 7 comprised by an adult pancreas.
 9. Mature β-cellshaving been genetically modified so that the processing of proinsulin toinsulin is substantially impaired.
 10. The β-cells of claim 9, whereinlevels of proinsulin convertase are substantially reduced in comparisonwith such levels in non-transgenic mice.
 11. The β-cells of claim 9comprised by an adult pancreas.
 12. A method of preventing or treatingtype II diabetes in a person by administering to said person apharmacologically effective amount of an agent that activates the FGFsignalling pathway in pancreatic β-cells promoting the formation ofproinsulin convertase and Glut2.
 13. A method of preventing or treatingtype II diabetes in a person by administering to said person a DNAfragment that allows expression of Ipf1 in pancreatic β-cells whichpromotes the formation of FGFR 1, proinsulin convertase and Glut2.